Anti-IL-17F Antibodies and Methods of Use Thereof

ABSTRACT

This invention provides fully human monoclonal antibodies that recognize IL-17F and/or the heterodimeric IL-17A/IL-17F complex, but do not recognize IL-17A. The invention further provides methods of using such monoclonal antibodies as a therapeutic, diagnostic, and prophylactic.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/175,512, filed May 5, 2009, the contents of which are herebyincorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “419USSeqListST25.txt,” which wascreated on Apr. 30, 2010 and is 47.3 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of monoclonalantibodies, e.g., fully human monoclonal antibodies, that recognizeIL-17F but do not recognize IL-17A, to monoclonal antibodies, e.g.,fully human monoclonal antibodies, that recognize the heterodimericIL-17A/IL-17F complex, and to methods of using the monoclonal antibodiesas therapeutics.

BACKGROUND OF THE INVENTION

IL-17F (also known as ML-1) is a member of the IL-17 family ofcytokines, which also includes the proteins IL-17A (also known as CTL-8,IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25). Both IL-17Aand IL-17F are secreted as disulfide linked homodimers which signalthrough the receptors IL-17R, IL-17RC, or a multimeric receptor complexcomposed of the IL-17R and IL-17RC. Both are also co-expressed on thesame T cell subsets (principally by the Th17 CD4⁺ T cells). IL-17A andIL-17F also interact and form a heterodimeric IL-17A/IL-17F complex.

Elevated levels of IL-17F and the IL-17A/IL-17F complex have beenassociated with a variety of inflammatory disorders and autoimmunediseases. Accordingly, there exists a need for therapies that neutralizethe biological activities of IL-17F.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies such as fully humanmonoclonal antibodies which specifically bind to IL-17F and/or theIL-17A/IL-17F heterodimeric complex, but do not specifically bind toIL-17A. IL-17F is typically expressed and biologically active as ahomodimeric protein. Thus, use of the term “IL-17F” and equivalentsthereof refers to the IL-17F homodimeric protein, except where otherwiseindicated. The antibodies of the invention are capable of modulating,e.g., blocking, inhibiting, reducing, antagonizing, neutralizing orotherwise interfering with IL-17F-mediated pro-inflammatory cytokineand/or chemokine production.

Exemplary monoclonal antibodies of the invention include, for example,the 5E12 antibody, the 41B10 antibody, the 11C5 antibody, the 21B10antibody, 1F1 antibody and 2E12 antibody. Alternatively, the monoclonalantibody is an antibody that binds to the same epitope as the 41B10antibody, the 11C5 antibody, the 21B10 antibody, 1F1 antibody, 2E12antibody, the 5D3 antibody, the 22F8 antibody, the 28B11 antibody, the41A4 antibody and the 43G6 antibody. These antibodies are respectivelyreferred to herein as “huIL17F” antibodies. huIL-17F antibodies includefully human monoclonal antibodies, as well as humanized monoclonalantibodies and chimeric antibodies.

Preferably, the fully human monoclonal antibody is selected from the11C5 antibody, the 21B10 antibody, 1F1 antibody, 2E12 antibody, 5D3antibody, 22F8 antibody, 28B11 antibody, 41A4 antibody and 43G6antibody. These antibodies exhibit higher affinity for IL-17F and/or theIL-17A/IL-17F heterodimeric complex than other antibodies that bindIL-17F and/or the IL-17A/IL-17F heterodimeric complex, such as, forexample, the 5E12 antibody and the 41B10 antibody. These antibodies arebetter inhibitors of at least one biological activity or function ofIL-17F than other antibodies that bind IL-17F and/or the IL-17A/IL-17Fheterodimeric complex, such as, for example, the 5E12 antibody and the41B10 antibody. For example, the 11C5 antibody, the 21B10 antibody, 1F1antibody, 2E12 antibody, 5D3 antibody, 22F8 antibody, 28B11 antibody,41A4 antibody and 43G6 antibody inhibit a biological activity and/orfunction of IL-17F to a greater degree than the 5E12 antibody and/or the41B10 antibody. In some embodiments, the 11C5 antibody, the 21B10antibody, 1F1 antibody, 2E12 antibody, 5D3 antibody, 22F8 antibody,28B11 antibody, 41A4 antibody and 43G6 antibody decrease production of apro-inflammatory cytokine in the presence of these antibodies to agreater degree than the decrease of pro-inflammatory cytokine productionin the presence of other antibodies that bind IL-17F and/or theIL-17A/IL-17F heterodimeric complex, such as, for example, the 5E12antibody and/or the 41B10 antibody. For example, the level ofpro-inflammatory cytokine (e.g., IL-6) production in the presence of the11C5 antibody, the 21B10 antibody, 1F1 antibody, 2E12 antibody, 5D3antibody, 22F8 antibody, 28B11 antibody, 41A4 antibody and 43G6 antibodyis greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 90%, 95%, 99%, or 100% lower than the level ofpro-inflammatory cytokine production other antibodies that bind IL-17Fand/or the IL-17A/IL-17F heterodimeric complex, such as, for example,the 5E12 antibody and/or the 41B10 antibody.

These antibodies show specificity for human IL-17F and/or the humanIL-17A/IL-17F heterodimeric complex, and they have been shown to inhibitIL-17F-mediated cytokine production. These antibodies have distinctspecificities. In some embodiments, the huIL-17F antibodies of theinvention specifically bind IL-17F, but do not specifically bind IL-17A.Preferably, the huIL-17 antibodies of the invention also specificallybind the IL-17F homodimer, but do not specifically bind the IL-17Ahomodimer. In some embodiments, the huIL-17F antibodies of the inventionspecifically bind the IL-17A/IL-17F heterodimeric complex, but do notspecifically bind IL-17A or the IL-17A homodimer. In some embodiments,the huIL-17F antibodies of the invention specifically bind IL-17F andthe IL-17A/IL-17F heterodimeric complex, but do not specifically bindIL-17A or the IL-17A homodimer. For example, the antibodies 11C5, 21B10,1F1, 2E12, 41B10, 5D3, 22F8, 28B11, 41A4 and 43G6 specifically bindIL-17F, and these antibodies do not bind IL-17A or the IL-17A homodimer.Preferably, the huIL-17F antibodies bind IL-17F and do not cross reactwith IL-17A or IL-17A homodimer. For example, 5E12, 11C5, 21B10, 1F1,2E12, 41B10, 5D3, 22F8, 28B11, 41A4 and 43G6 bind IL-17F but do notcross react with IL-17A.

The fully human antibodies of the invention contain a heavy chainvariable region having the amino acid sequence of SEQ ID NOS: 10, 14,18, 22, 26, 30, 34, 38 and 42. The human antibodies of the inventioncontain a light chain variable region having the amino acid sequence ofSEQ ID NOS: 12, 16, 20, 24, 28, 32, 36, 40 and 44. The heavy chain CDRsinclude a VH CDR1 region comprising an amino acid sequence at least 90%,92%, 95%, 97% 98%, 99% or more identical to a sequence selected from thegroup consisting of SEQ ID NOS: 45, 48, 51, 56, 59, 64, 67 and 70; a VHCDR2 region comprising an amino acid sequence at least 90%, 92%, 95%,97% 98%, 99% or more identical to a sequence selected from the groupconsisting of SEQ ID NOS: 46, 49, 52, 54, 57, 60, 62, 65, 68, 71 and 73;and a VH CDR3 region comprising an amino acid sequence at least 90%,92%, 95%, 97% 98%, 99% or more identical to a sequence selected from thegroup consisting of SEQ ID NOS: 47, 50, 53, 55, 58, 61, 63, 66, 69 and72. The three light chain CDRs include a VL CDR1 region comprising anamino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or moreidentical to a sequence selected from the group consisting of SEQ IDNOS: 74, 77, 80, 85, 88, 91 and 94; a VL CDR2 region comprising an aminoacid sequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical toa sequence selected from the group consisting of SEQ ID NOS: 75, 78, 81,83, 86, 89, 92 and 96; and a VL CDR3 region comprising an amino acidsequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to asequence selected from the group consisting of SEQ ID NOS: 76, 79, 82,84, 87, 90, 93, 95, 97, 98 and 99.

Preferably, the heavy chain CDRs include a VH CDR1 region comprising anamino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or moreidentical to a sequence selected from the group consisting of SEQ IDNOS: 45, 48, 51, 64, 67 and 70; a VH CDR2 region comprising an aminoacid sequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical toa sequence selected from the group consisting of SEQ ID NOS: 46, 49, 52,54, 62, 65, 68, 71 and 73; and a VH CDR3 region comprising an amino acidsequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to asequence selected from the group consisting of SEQ ID NOS: 47, 50, 53,55, 63, 66, 69 and 72. The three light chain CDRs include a VL CDR1region comprising an amino acid sequence at least 90%, 92%, 95%, 97%98%, 99% or more identical to a sequence selected from the groupconsisting of SEQ ID NOS: 74, 77, 80, 85, 91 and 94; a VL CDR2 regioncomprising an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99%or more identical to a sequence selected from the group consisting ofSEQ ID NOS: 75, 78, 81, 83, 86, 92 and 96; and a VL CDR3 regioncomprising an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99%or more identical to a sequence selected from the group consisting ofSEQ ID NOS: 76, 79, 82, 84, 93, 95, 97, 98 and 99, provided that whenthe VL CDR1 comprises an amino acid sequence that is at least 90%, 92%,95%, 97% 98%, 99% or more identical to the sequence of SEQ ID NO:85, theVL CDR2 comprises an amino acid sequence that is at least 90%, 92%, 95%,97% 98%, 99% or more identical to the sequence of SEQ ID NO: 86 and theVL CDR3 comprises an amino acid sequence that is at least 90%, 92%, 95%,97% 98%, 99% or more identical to the sequence of SEQ ID NO:98, andprovided that when the VL CDR2 comprises an amino acid sequence that isat least 90%, 92%, 95%, 97% 98%, 99% or more identical to the sequenceof SEQ ID NO:86, the VL CDR1 comprises an amino acid sequence that is atleast 90%, 92%, 95%, 97% 98%, 99% or more identical to the sequence ofSEQ ID NO: 85 and the VL CDR3 comprises an amino acid sequence that isat least 90%, 92%, 95%, 97% 98%, 99% or more identical to the sequenceof SEQ ID NO:98.

Antibodies of the invention immunospecifically bind IL-17F wherein theantibody binds to an epitope that includes one or more amino acidresidues on human IL-17F. In some embodiments, antibodies of theinvention also specifically bind the heterodimeric IL-17A/IL-17F complexbut not IL-17A, wherein the antibody binds to an epitope that includesone or more amino acid residues on human IL-17F.

Antibodies of the invention also include fully human antibodies thatspecifically bind IL-17F and/or the heterodimeric IL-17A/IL-17F complexwherein the antibody exhibits greater than 50% inhibition ofIL-17F-mediated pro-inflammatory cytokine production in vitro. Forexample, antibodies of the invention exhibit greater than 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% inhibition of IL-6secretion by IL-17 stimulated cells. As used herein, the term“pro-inflammatory cytokine” refers to those immunoregulatory cytokinesthat promote inflammation and/or are associated with inflammation.Pro-inflammatory cytokines and chemokines include, for example, IL-6,IL-8, G-CSF, and GM-CSF. Pro-inflammatory chemokines include, forexample, GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES,Eotaxin, SDF-1, and MIP3a.

The present invention also provides methods of treating or preventingpathologies associated with aberrant IL-17F activity (e.g., aberrantpro-inflammatory cytokine production, such as for example aberrant IL-6production), or alleviating a symptom associated with such pathologies,by administering a monoclonal antibody of the invention (e.g., fullyhuman monoclonal antibody) to a subject in which such treatment orprevention is desired. The subject to be treated is, e.g., human. Themonoclonal antibody is administered in an amount sufficient to treat,prevent or alleviate a symptom associated with the pathology. The amountof monoclonal antibody sufficient to treat or prevent the pathology inthe subject is, for example, an amount that is sufficient to reduceIL-17F signaling (e.g., IL-17F-induced production of one or morepro-inflammatory cytokines such as e.g., IL-6). As used herein, the term“reduced” refers to a decreased production of a pro-inflammatorycytokine in the presence of a monoclonal antibody of the invention,wherein the production is, for example, local pro-inflammatory cytokineproduction (e.g., at a site of inflamed tissue) or systemicpro-inflammatory cytokine production. IL-17F signaling (e.g.,IL-17F-induced pro-inflammatory cytokine such as IL-6) is decreased whenthe level of pro-inflammatory cytokine (e.g., IL-6) production in thepresence of a monoclonal antibody of the invention is greater than orequal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%,99%, or 100% lower than a control level of pro-inflammatory cytokineproduction (i.e., the level of pro-inflammatory cytokine production inthe absence of the monoclonal antibody). Level of pro-inflammatorycytokine production (e.g., IL-6) is measured, e.g., using theIL-17F-stimulated Mouse Embryonic Fibroblasts (MEF) cellular assaysdescribed herein. Those skilled in the art will appreciate that thelevel of pro-inflammatory cytokine production can be measured using avariety of assays, including, for example, commercially available ELISAkits. Pathologies treated and/or prevented using the monoclonalantibodies of the invention (e.g., fully human monoclonal antibody)include, for example, acute inflammation, chronic inflammation (e.g.,chronic inflammation associated with allergic conditions and asthma,chronic inflammation associated with arthritic conditions), autoimmunediseases (e.g., Crohn's disease, multiple sclerosis, rheumatoidarthritis and other autoimmune arthritic conditions), inflammatory boweldisease, and transplant rejection.

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

The present invention also provides soluble IL-17F proteins, methods forexpressing IL-17F proteins, and methods for purifying such proteins in asoluble form.

In some embodiments, the pathology to be treated is one or moreautoimmune diseases, inflammatory disorders or cancers. For example,without limitation, the pathology is rheumatoid arthritis and otherarthritic conditions, Crohn's disease, psoriasis, multiple sclerosischronic obstructive pulmonary disease and/or asthma, cancer andangiogenesis.

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

One skilled in the art will appreciate that the antibodies of theinvention have a variety of uses. For example, the proteins of theinvention are used as therapeutic agents to prevent the activation ofIL-17F receptor in disorders such as, for example, rheumatoid arthritis,Crohn's disease, psoriasis, multiple sclerosis chronic obstructivepulmonary disease, angiogenesis, asthma and cancer. The antibodies ofthe invention are also used as reagents in diagnostic kits or asdiagnostic tools, or these antibodies can be used in competition assaysto generate therapeutic reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that depicts the progression of clinical scoring ofcollagen-induced arthritis in mice using standard arthritis scoringmethods.

FIGS. 2A-2F are a series of graphs that depict various cytokine serumlevels in mice immunized with bovine collagen type II as determined attermination (day 22). FIG. 2A depicts the detected serum level of tumornecrosis factor alpha (TNF-α); FIG. 2B depicts the detected serum levelof interleukin 6 (IL-6); FIG. 2C depicts the detected serum level ofinterferon gamma (IFN-γ); FIG. 2D depicts the detected serum level ofinterleukin 1 alpha (IL-1α); FIG. 2E depicts the detected serum level ofmonocyte chemotactic protein (MCP-1); and FIG. 2F depicts the detectedserum level of the interleukin 12/interleukin 23 (IL-12/IL-23)heterodimeric complex.

DETAILED DESCRIPTION

The present invention provides monoclonal antibodies that specificallybind IL-17F. The invention further provides monoclonal antibodies thatspecifically bind IL-17F and the heterodimeric IL-17A/IL-17F complex(also referred to herein as the IL-17A/IL-17F heterodimer). The antibodyis e.g., a fully human monoclonal antibody.

Antibodies of the invention specifically bind IL-17F but not IL-17A,wherein the antibody binds to an epitope that includes one or more aminoacid residues of human IL-17F. In some embodiments, antibodies of theinvention specifically bind IL-17F and the heterodimeric IL-17A/IL-17Fcomplex but not IL-17A or the IL-17A homodimer, wherein the antibodybinds to an epitope that includes one or more amino acid residues ofhuman IL-17F.

The antibodies of the present invention bind to an IL-17F epitope withan equilibrium binding constant (K_(d)) of ≦1 μM, e.g., ≦100 nM,preferably ≦10 nM, and more preferably ≦1 nM. For example, the huIL-17Fantibodies provided herein exhibit a K_(d) in the range approximatelybetween ≦1 nM to about 1 pM.

The crystal structure of IL-17F reveals that the protein adopts acysteine knot fold, suggesting a relationship to the cysteine knotsuperfamily of proteins. However, the cysteine knot motif of IL-17F onlyutilizes four cysteines instead of the classical six cysteines to formthe knot. Like other members of the cysteine knot family, IL-17F alsoexists as a heterodimer with IL-17A. The IL-17A/IL-17F heterodimer isbelieved to signal through IL-17R and/or the multimeric IL-17R/IL-17RCcomplex. Recent evidence has shown that the same cysteine residues thatare utilized in forming the IL-17A/IL-17F heterodimer are the samecysteines utilized in the IL-17F homodimer formation. This data suggeststhat the receptor for the IL-17F homodimer or IL-17A/IL-17F heterodimermay bind to the conserved cysteine residues at the dimer interface, likeother proteins in the cysteine knot family.

Numerous immune regulatory functions have been reported for the IL-17family of cytokines, presumably due to their induction of many immunesignaling molecules. IL-17A, expressed as the IL-17A homodimer, andIL-17F, expressed as the IL-17F homodimer, share very similar biologicalfunctions in some cases. Both promote secretion of pro-inflammatorycytokines (e.g., IL-6, IL-8, G-CSF, and GM-CSF), chemokines (e.g.,GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin,SDF-1, and MIP3a) and prostaglandins (e.g., PGE₂) from a wide variety ofcells including fibroblasts, keratinocytes, macrophages, epithelialcells and endothelial cells. Both have also been shown to regulatecartilage matrix turnover. IL-17F homodimer also have biologicalfunctions distinct from IL-17A homodimer such as the ability tostimulate proliferation and activation of T cells and peripheral bloodmononuclear cells (PBMCs), and to inhibit angiogenesis.

The huIL17F antibodies of the invention serve to modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere with thebiological activity of IL-17F. Biological activities of IL-17F include,for example, binding to IL-17R, IL-17RC and/or the multimericIL-17R/IL-17RC receptor complex, and the induction of cytokine and/orchemokine expression (e.g., IL-6, IL-8, G-CSF, GM-CSF, GRO-α, GRO-b,LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF-1, andMIP3a) in target cells. For example, the huIL-17F antibodies completelyor partially inhibit IL-17F biological activity by partially orcompletely modulating, blocking, inhibiting, reducing antagonizing,neutralizing, or otherwise interfering with the binding of IL-17F to itsreceptor, or otherwise partially or completely modulating, blocking,inhibiting, reducing, antagonizing, neutralizing IL-17F signalingactivity.

The hulL-17F antibodies are considered to completely modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withIL-17F biological activity when the level of IL-17F activity in thepresence of the huIL-17F antibody is decreased by at least 95%, e.g., by96%, 97%, 98%, 99% or 100% as compared to the level of IL-17F activityin the absence of binding with an huIL-17F antibody described herein.The huIL-17F antibodies are considered to partially modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withIL-17F activity when the level of IL-17F activity in the presence of thehuIL-17F antibody is decreased by less than 95%, e.g., 10%, 20%, 25%,30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level ofIL-17F activity in the absence of binding with an huIL-17F antibodydescribed herein.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms Interleukin-17A, IL-17A, IL17A, IL-17, IL17,CTLA8, CTLA-8, Cytotoxic T-lymphocyte-associated antigen 8 andInterleukin-17A precursor are synonymous and may be usedinterchangeably. Each of these terms refers to the homodimeric protein,except where otherwise indicated.

As used herein, the terms Interleukin-17F, IL-17F, IL17F, ML-1, ML1,Interleukin-24, IL-24, IL24 and Interleukin-17F precursor are synonymousand may be used interchangeably. Each of these terms refers to theIL-17F homodimeric protein, except where otherwise indicated.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≦1 μM;e.g., ≦100 nM, preferably ≦10 nM and more preferably ≦1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to IL-17F and/or the IL-17A/IL-17Fheterodimer, when the equilibrium binding constant (K_(d)) is ≦1 μM,preferably ≦100 nM, more preferably ≦10 nM, and most preferably ≦100 pMto about 1 pM, as measured by assays such as radioligand binding assaysor similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules presented in SEQ IDNOS: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37 and 41, and nucleic acidmolecules encoding the light chain immunoglobulin molecules representedin SEQ ID NOS: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39 and 43.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules represented in SEQ ID NOS: 2, 6, 10, 14,18, 22, 26, 30, 34, 38 and 42, and the light chain immunoglobulinmolecules represented in SEQ ID NOS: 4, 8, 12, 16, 20, 24, 28, 32, 36,40 and 44, as well as antibody molecules formed by combinationscomprising the heavy chain immunoglobulin molecules with light chainimmunoglobulin molecules, such as kappa light chain immunoglobulinmolecules, and vice versa, as well as fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers D-amino acids) of the twentyconventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long' morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto IL-17F alone or IL-17A/IL-17F heterodimer (i.e., complex), undersuitable binding conditions. Typically, polypeptide analogs comprise aconservative amino acid substitution (or addition or deletion) withrespect to the naturally-occurring sequence. Analogs typically are atleast 20 amino acids long, preferably at least 50 amino acids long orlonger, and can often be as long as a full-length naturally-occurringpolypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

huIL-17F Antibodies

Monoclonal antibodies of the invention (e.g., fully human monoclonalantibodies) bind IL-17F, and in some embodiments, the IL-17A/IL-17Fheterodimeric complex, but do not bind IL-17A or the IL-17A homodimer.These monoclonal antibodies have the ability to inhibit IL-17F-inducedproinflammatory cytokine production (e.g., IL-6). Inhibition isdetermined, for example, the IL-17F stimulated mouse embryonicfibroblast (MEF) cellular assays described herein.

Exemplary antibodies of the invention include, for example, the 5E12antibody, the 41B10 antibody, the 11C5 antibody, the 21B10 antibody, the1F1 antibody, the 2E12 antibody, the 5D3 antibody, the 22F8 antibody,the 28B11 antibody, the 41A4 antibody and the 43G6 antibody describedherein. These antibodies show specificity for human IL-17F and/or theheterodimeric IL-17A/IL-17F complex, and they have been shown to inhibithuman IL-17F induction of the pro-inflammatory cytokine IL-6 in vitro.

Each of the huIL-17F monoclonal antibodies described herein includes aheavy chain variable region (VH) and a light chain variable region (VL),as shown in the amino acid and corresponding nucleic acid sequenceslisted below.

The 5E12 antibody includes a heavy chain variable region (SEQ ID NO:2)encoded by the nucleic acid sequence shown in SEQ ID NO:1, and a lightchain variable region (SEQ ID NO:4) encoded by the nucleic acid sequenceshown in SEQ ID NO:3.

>5E12 VH nucleic acid sequence (SEQ ID NO: 1)CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTACCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGAACTGTATATCAGTGACTGGGACTCCTACTCCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA >5E12VH amino acid sequence (SEQ ID NO: 2)QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKELYISDWDSYSYGMDVWGQGTTVTVSS >5E12 VLnucleic acid sequence (SEQ ID NO: 3)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >5E12 VL amino acid sequence (SEQ ID NO: 4)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFGGGTKVEIK

The 41B10 antibody includes a heavy chain variable region (SEQ ID NO:6)encoded by the nucleic acid sequence shown in SEQ ID NO:5, and a lightchain variable region (SEQ ID NO:8) encoded by the nucleic acid sequenceshown in SEQ ID NO:7.

>41B10 VH nucleic acid sequence (SEQ ID NO: 5)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGTTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACCCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTATATTACTGTACCACATCGTATAGCAGTTACTGGTTCCCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA >41B10 VHamino acid sequence (SEQ ID NO: 6)EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYVAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTSYSSYWFPYYFDYWGQGTLVTVSS >41B10 VLnucleic acid sequence (SEQ ID NO: 7)GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA >41B10 VL amino acid sequence (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPITFGQGTRLEIK

The 11C5 antibody includes a heavy chain variable region (SEQ ID NO:10)encoded by the nucleic acid sequence shown in SEQ ID NO:9, and a lightchain variable region (SEQ ID NO:12) encoded by the nucleic acidsequence shown in SEQ ID NO:11. The amino acid sequence of the lightchain variable region for the 11C5 antibody include mutations at the 5′end to convert the residues to the residues found in the correspondinghuman germline sequence. The non-mutated version of the light chainvariable region amino acid sequence for the 11C5 antibody is shown inSEQ ID NO: 102, and the non-mutated version of the light chain variableregion nucleic acid sequence for the 11C5 antibody is shown in SEQ IDNO: 103.

>11C5 VH Nucleic acid sequence (SEQ ID NO: 9)CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCATCTATTATTTGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGGACAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATGACCAGGGACCCGTCCACGAACACAGTCTACATGGAACTGAGCAGCCTGACATCTGAGGACGCGGCCGTGTATTACTGTGCGAGAGGGGAATTTAGCAGTGGCTGGCTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >11C5 VH Aminoacid sequence (SEQ ID NO: 10)QVQLVQSGAEVKKPGASVKVSCKASGYTFTIYYLHWVRQAPGQGLEWMGIINPSGGRTNYAQKFQGRVTMTRDPSTNTVYMELSSLTSEDAAVYYCARGEFSSGWLDYWGQGTTVTVSS >11C5 VLNucleic acid sequence (SEQ ID NO: 11)GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >11C5 VL Amino acid sequence (SEQ ID NO: 12)DIQMTQPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK >Non-mutated 11C5 VLNucleic acid sequence (SEQ ID NO: 102)GACATCGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >Non-mutated 11C5 VL Amino acid sequence (SEQ IDNO: 103) DIVMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK

The 21B10 antibody includes a heavy chain variable region (SEQ ID NO:14)encoded by the nucleic acid sequence shown in SEQ ID NO:13, and a lightchain variable region (SEQ ID NO:16) encoded by the nucleic acidsequence shown in SEQ ID NO:15.

>21B10 VH Nucleic acid sequence (SEQ ID NO: 13)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTGGTGGTAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGCTATGTTTCGGGGACCTATTACAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >21B10 VH Amino acid sequence (SEQ ID NO: 14)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISGGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREGYVSGTYYNYYYGMDVWGQGTTVTVSS >21B10 VL Nucleic acid sequence (SEQ ID NO: 15)GAAATTGTGTTGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCACCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGTCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >21B10 VL amino acid sequence (SEQ ID NO: 16)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAPNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWSLTFGGGTKVEIK

The 1F1 antibody includes a heavy chain variable region (SEQ ID NO:18)encoded by the nucleic acid sequence shown in SEQ ID NO:17, and a lightchain variable region (SEQ ID NO 20) encoded by the nucleic acidsequence shown in SEQ ID NO:19. The amino acid sequence of the lightchain variable region for the 1F1 antibody include mutations at the 5′end to convert the residues to the residues found in the correspondinghuman germline sequence. The non-mutated version of the light chainvariable region amino acid sequence for the 1F1 antibody is shown in SEQID NO: 104, and the non-mutated version of the light chain variableregion nucleic acid sequence for the 1F1 antibody is shown in SEQ ID NO:105.

>1F1 VH Nucleic acid sequence (SEQ ID NO: 17)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGTCATGAGCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTCGTGGTGGTAACACATTCTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGATCGGCGTATAGCAGCAGGTAGTTTTGACTATTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA >1F1 VHAmino acid sequence (SEQ ID NO: 18)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQVPGKGLEWVSAISGRGGNTFYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCAKDDRRIAAGSFDYWGQGTTVTVSS >1F1 VLNucleic acid sequence (SEQ ID NO: 19)GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGTCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >1F1 VL Amino acid sequence (SEQ ID NO: 20)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDVSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK >Non-mutated 1F1 VLNucleic acid sequence (SEQ ID NO: 104)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGTCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >Non-mutated 1F1 VL Amino acid sequence (SEQ IDNO: 105) DIQMTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDVSSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

The 2E12 antibody includes a heavy chain variable region (SEQ ID NO:22)encoded by the nucleic acid sequence shown in SEQ ID NO:21, and a lightchain variable region (SEQ ID NO 24) encoded by the nucleic acidsequence shown in SEQ ID NO:23.

>2E12 VH Nucleic acid sequence (SEQ ID NO: 21)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCGGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTAGTAGTAGTAGTGCCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGCTATGCTTCGGGGAGGTATTACAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >2E12 VH Amino acid sequence (SEQ ID NO: 22)EVQLVESGGGLVQRGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWISYISSSSSAIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREGYASGRYYNYYYGMDVWGQGTTVTVSS >2E12 VL Nucleic acid sequence (SEQ ID NO: 23)GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTGGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGTCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCGGTTTATTACTGTCAGCAGCGTAGCAGCTGGTCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >2E12 VL Amino acid sequence (SEQ ID NO: 24)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSVSGSGTDFTLTISSLEPEDFAVYYCQQRSSWSLTFGGGTKVEIK

The 5D3 antibody includes a heavy chain variable region (SEQ ID NO:26)encoded by the nucleic acid sequence shown in SEQ ID NO:25, and a lightchain variable region (SEQ ID NO:28) encoded by the nucleic acidsequence shown in SEQ ID NO:27.

>5D3 VH Nucleic acid sequence (SEQ ID NO: 25)GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCAGAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATGGTCTACGCTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >5D3 VH Amino acidsequence (SEQ ID NO: 26)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSRGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDMVYALDVWGQGTTVTVSS >5D3 VLNucleic acid sequence (SEQ ID NO: 27)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTTTTAGCGGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATACATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTACGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA >5D3 VL Nucleic acid sequence (SEQ ID NO: 28)EIVLTQSPGTLSLSPGERATLSCRASQSFSGSYLAWYQQKPGQAPRLLIYDTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGTWTFGQGTKVEIK

The 22F8 antibody includes a heavy chain variable region (SEQ ID NO:30)encoded by the nucleic acid sequence shown in SEQ ID NO:29, and a lightchain variable region (SEQ ID NO:32) encoded by the nucleic acidsequence shown in SEQ ID NO:31.

>22F8 VH Nucleic acid sequence (SEQ ID NO: 29)GAGGTGCAGTTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTCGTGGTGGTAGCATATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGAGGAGGCTACCTGGGACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >22F8 VH Aminoacid sequence (SEQ ID NO: 30)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSTISGRGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEATWDFDYWGQGTTVTVSS >22F8 VLNucleic acid sequence (SEQ ID NO: 31)GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTTCCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA >22F8 VL Amino acid sequence (SEQ ID NO: 32)EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWFQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

The 28B11 antibody includes a heavy chain variable region (SEQ ID NO:34)encoded by the nucleic acid sequence shown in SEQ ID NO:33, and a lightchain variable region (SEQ ID NO:36) encoded by the nucleic acidsequence shown in SEQ ID NO:35.

>28B11 VH Nucleic acid sequence (SEQ ID NO: 33)CAGGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTACTACATGACCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTAGTACTGGTGGTAACATCTACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCCAGAATTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGGGTGTAATAATCTCAACTGCTATGTTTGACTATTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >28B11 VH Amino acid sequence (SEQ ID NO: 34)QVQLVESGGGLVKPGGSLRLSCAASGFTFSNYYMTWIRQAPGKGLEWISYISSTGGNIYYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARDGGVIISTAMFDYWGQGTTVTV SS >28B11VL Nucleic acid sequence (SEQ ID NO: 35)GCCATCCAGTTGACCCAGTCTCCCTCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGCTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >28B11 VL Amino acid sequence (SEQ ID NO: 36)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRLSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK

The 41A4 antibody includes a heavy chain variable region (SEQ ID NO:38)encoded by the nucleic acid sequence shown in SEQ ID NO:37, and a lightchain variable region (SEQ ID NO:40) encoded by the nucleic acidsequence shown in SEQ ID NO:39.

>41A4 VH Nucleic acid sequence (SEQ ID NO: 37)GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTGTGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCGTATACTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAATTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTACAAAAGAAAAATACAACTGGAACGACGAGGGGGAATACTTCTACGGAATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >41A4 VH Amino acid sequence (SEQ ID NO: 38)EVQLVESGGGLVQPGRSLRLSCAASGFTFDDCAMHWVRQAPGKGLEWVSGISWNSGSVYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKEKYNWNDEGEYFYGMDVWGQGTTVTVSS >41A4 VL Nucleic acid sequence (SEQ ID NO: 39)GAAATTGTGTTGACACAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >41A4 VL Amino acid sequence (SEQ ID NO: 40)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSFGGGTKVEIK

The 43G6 antibody includes a heavy chain variable region (SEQ ID NO:42)encoded by the nucleic acid sequence shown in SEQ ID NO:41, and a lightchain variable region (SEQ ID NO:44) encoded by the nucleic acidsequence shown in SEQ ID NO:43.

>43G6 VH3 Nucleic acid sequence (SEQ ID NO: 41)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTGGTAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGAGAGAGGGCTATGTTTCGGGGACCTATTACAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA >43G6 VH3 Amino acid sequence (SEQ ID NO: 42)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSGSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREGYVSGTYYNYYYGMDVWGQGTTVTVSS >43G6 VL3 Nucleic acid sequence (SEQ ID NO: 43)GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGTTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCACCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGTCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA >43G6 VL3 Amino acid sequence (SEQ ID NO: 44)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAPNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWSLTFGGGTKVEIK

huIL-17F antibodies of the invention also include, e.g., the heavy chaincomplementarity determining regions (VH CDRs) shown in Table 1, thelight chain CDRs (VL CDRs) shown in Table 2, and combinations thereof.

TABLE 1 VH CDR sequences from antibody clones that bind and neutralizeIL-17F biological activity Clone ID Heavy CDR1 Heavy CDR2 Heavy CDR3Gene family 11C5 IYYLH IINPSGGRTNYAQFQG GEFSSGWLDY IGHV1-46 (SEQ ID NO:45) (SEQ ID NO: 46) (SEQ ID NO: 47) 21B10 SYSMN YISGGSSTIYYADSVKGEGYVSGTYYNYYYGMDV IGHV3-48 (SEQ ID NO: 48) (SEQ ID NO: 49) (SEQ ID NO:50) 1F1 SYVMS AISGRGGNTFYADSVKG DDRRIAAGSFDY IGHV3-23 (SEQ ID NO: 51)(SEQ ID NO: 52) (SEQ ID NO: 53) 2E12 SYSMN YISSSSSAIYYADSVKGEGYASGRYYNYYYGMDV IGHV3-48 (SEQ ID NO: 48) (SEQ ID NO: 54) (SEQ ID NO:55) 5E12 DYAMH GISWNSGTIGYADSVKG ELYISDWDSYSYGMDV IGHV3-9 IgG4 (SEQ IDNO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58) 41B10 NAWMS RIKSKTDGGTTDYVAPVKGSYSSYWFPYYFDY IGHV3-15 IgG4 (SEQ ID NO: 59) (SEQ ID NO: 60) (SEQ ID NO:61) 5D3 DYAMH GISWNSGSRGYADSVKG DMVYALDV IGHV3-9 (SEQ ID NO: 56) (SEQ IDNO: 62) (SEQ ID NO: 63) 22F8 SYAMN TISGRGGSIYYADSVKG EEATWDFDY IGHV3-23(SEQ ID NO: 64) (SEQ ID NO: 65) (SEQ ID NO: 66) 28B11 NYYMTYISSTGGNIYYADSVKG DGGVIISTAMFDY IGHV3-11 (SEQ ID NO: 67) (SEQ ID NO: 68)(SEQ ID NO: 69) 41A4 DCAMH GISWNSGSVYYADSVKG EKYNWNDEGEYFYGMDV IGHV3-9(SEQ ID NO: 70) (SEQ ID NO: 71) (SEQ ID NO: 72) 43G6 SYSMNYISSGSSTIYYADSVKG EGYVSGTYYNYYYGMDV IGHV3-48 (SEQ ID NO: 48) (SEQ ID NO:73) (SEQ ID NO: 50)

TABLE 2 VL CDR sequences from antibody clones that bind and neutralizeIL-17F Clone ID Light CDR1 Light CDR2 Light CDR3 Gene family 11C5RASQGISSWLA AASSLQS ANSFPLT IGKV1-12 (SEQ ID NO: 74) (SEQ ID NO: 75)(SEQ ID NO: 76) IGKJ4 21B10 RASQSVSSYLA DAPNRAT RSNWSLT IGKV3-11 (SEQ IDNO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) IGKJ4 1F1 RASQGISSALA DVSSLESFNSYPLT IGKV1D-13 (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 82) IGKJ42E12 RASQSVSSYLA DASNRAT RSSWSLT IGKV3-11 (SEQ ID NO: 77) (SEQ ID NO:83) (SEQ ID NO: 84) IGKJ4 5E12 RASQSVSSSYLA GASSRAT QQYGSSP IGKV3-20(SEQ ID NO: 85) (SEQ ID NO: 86) (SEQ ID NO: 87) 41B10 RASQGISSWLAAASSLQS QQYNSYPIT IGKV1D-16 (SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO:90) 5D3 RASQSFSGSYLA DTSSRAT QQYGTWT IGKV3-20 (SEQ ID NO: 91) (SEQ IDNO: 92) (SEQ ID NO: 93) 22F8 RASQSVSSFLA DASNRAT QQRSNWPPT IGKV3-11 (SEQID NO: 94) (SEQ ID NO: 83) (SEQ ID NO: 95) 28B11 RASQGISSALA DASSLESQQFNSYPLT IGKV1D-13 (SEQ ID NO: 80) (SEQ ID NO: 96) (SEQ ID NO: 97) 41A4RASQSVSSSYLA GASSRAT QQYGSS IGKV3-20 (SEQ ID NO: 85) (SEQ ID NO: 86)(SEQ ID NO: 98) 43G6 RASQSVSSYLA DAPNRAT QQRSNWSLT IGKV3-11 (SEQ ID NO:77) (SEQ ID NO: 78) (SEQ ID NO: 99)

The amino acids encompassing the complementarity determining regions

(CDR) are as defined by E. A. Kabat et al. (See Kabat, E A, et al.,Sequences of Protein of immunological interest, Fifth Edition, USDepartment of Health and Human Services, US Government Printing Office(1991)).

Also included in the invention are antibodies that bind to the sameepitope as the antibodies described herein. For example, antibodies ofthe invention specifically bind to IL-17F and/or the IL-17A/IL-17Fheterodimeric complex, wherein the antibody binds to an epitope thatincludes one or more amino acid residues on human IL-17F (Accession No.AAH70124). In some embodiments, antibodies of the invention specificallybind IL-17F and the heterodimeric IL-17A/IL-17F complex, wherein theantibody binds to an epitope on human IL-17F (e.g., Accession No.AAH70124).

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., fully human monoclonal antibody) has the same specificity as amonoclonal antibody of the invention (e.g., clones 5E12, 41B10, 11C5,21B10, 1F1, 2E12, 5D3, 22F8, 28B11, 41A4 and 43G6) by ascertainingwhether the former prevents the latter from binding to IL-17F and/or theheterodimeric IL-17A/IL-17F complex. If the monoclonal antibody beingtested competes with the monoclonal antibody of the invention, as shownby a decrease in binding by the monoclonal antibody of the invention,then the two monoclonal antibodies bind to the same, or a closelyrelated, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with solubleIL-17F and/or soluble heterodimeric IL-17A/IL-17F complex proteins andthen add the monoclonal antibody being tested to determine if themonoclonal antibody being tested is inhibited in its ability to bindIL-17F and/or the heterodimeric IL-17A/IL-17F complex. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

Screening of monoclonal antibodies of the invention, can be also carriedout, e.g., by measuring IL-17F-induced cytokine and/or chemokineproduction (e.g., IL-6, IL-8, G-CSF, GM-CSF, GRO-α, GRO-b, LIX, GCP-2,MIG, IP10, I-TAC, and MCP-1, RANTES, Eotaxin, SDF-1, and MIP3a) anddetermining whether the test monoclonal antibody is able to modulate,block, inhibit, reduce, antagonize, neutralize or otherwise interferewith IL-17F-induced cytokine and/or chemokine production.

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against the IL-17F, or againstderivatives, fragments, analogs homologs or orthologs thereof (See,e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporatedherein by reference). Fully human antibodies are antibody molecules inwhich the entire sequence of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies are prepared, for example, using the procedures described inthe Examples provided below. Human monoclonal antibodies can be alsoprepared by using the trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBVhybridoma technique to produce human monoclonal antibodies (see Cole, etal., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may beproduced by using human hybridomas (see Cote, et al., 1983. Proc NatlAcad Sci USA 80: 2026-2030) or by transforming human B-cells withEpstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The antibodies of the invention (e.g., 5E12, 41B10, 11C5, 21B10, 1F1,2E12, 5D3, 22F8, 28B11, 41A4 and 43G6) are fully human monoclonalantibodies. Monoclonal antibodies that modulate, block, inhibit, reduce,antagonize, neutralize or otherwise interfere with pro-inflammatorycytokine production mediated by IL-17F are generated, e.g., byimmunizing an animal with IL-17F such as, for example, murine, rat orhuman IL-17F or an immunogenic fragment, derivative or variant thereof.Alternatively, the animal is immunized with cells transfected with avector containing a nucleic acid molecule encoding IL-17F, such thatIL-17F is expressed and associated with the surface of the transfectedcells. Alternatively, the antibodies are obtained by screening a librarythat contains antibody or antigen binding domain sequences for bindingto IL-17F. This library is prepared, e.g., in bacteriophage as proteinor peptide fusions to a bacteriophage coat protein that is expressed onthe surface of assembled phage particles and the encoding DNA sequencescontained within the phage particles (i.e., “phage displayed library”).Hybridomas resulting from myeloma/B cell fusions are then screened forreactivity to IL-17F.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

Monoclonal antibodies of the invention include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

A huIL-17F antibody is generated, for example, using the proceduresdescribed in the Examples provided below.

In other, alternative methods, a huIL-17F antibody is developed, forexample, using phage-display methods using antibodies containing onlyhuman sequences. Such approaches are well-known in the art, e.g., inWO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated byreference. In this approach, a combinatorial library of phage carryingrandom pairs of light and heavy chains are screened using natural orrecombinant source of IL-17F or fragments thereof. In another approach,a huIL-17F antibody can be produced by a process wherein at least onestep of the process includes immunizing a transgenic, non-human animalwith human IL-17F protein. In this approach, some of the endogenousheavy and/or kappa light chain loci of this xenogenic non-human animalhave been disabled and are incapable of the rearrangement required togenerate genes encoding immunoglobulins in response to an antigen. Inaddition, at least one human heavy chain locus and at least one humanlight chain locus have been stably transfected into the animal. Thus, inresponse to an administered antigen, the human loci rearrange to providegenes encoding human variable regions immunospecific for the antigen.Upon immunization, therefore, the xenomouse produces B-cells thatsecrete fully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181and No. 6,150,584, which is hereby incorporated by reference in itsentirety. This general strategy was demonstrated in connection withgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963, 6,150,584, 6, 114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more D_(H) genes, one or more J_(H) genes, a mu constant region,and a second constant region (preferably a gamma constant region) areformed into a construct for insertion into an animal. See e.g., U.S.Pat. Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205;5,625,825;5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429;5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and6,255,458; and European Patent No. 0 546 073 B1; and InternationalPatent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO97/13852, and WO 98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a immune variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against IL-17F and/or the heterodimeric IL-17A/IL-17F complexin order to vitiate or otherwise mitigate concerns and/or effects ofHAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92/102190 and U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG3 and IgG4. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

Antibody fragments, such as Fv, F(ab)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as willbe appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPlückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to IL-17F expressingcells expressing cells, IL-17F itself, forms of IL-17F epitopes orpeptides thereof, and expression libraries thereto (See e.g., U.S. Pat.No. 5,703,057) which can thereafter be screened as described above forthe activities described herein.

The huIL-17F antibodies of the invention can be expressed by a vectorcontaining a DNA segment encoding the single chain antibody describedabove.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA.gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci. USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofIL-17F in a sample. The antibody can also be used to try to bind to anddisrupt IL-17F related signaling.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)anti-IL-17F fragments, single chain anti-IL-17F antibodies, bispecificanti-IL-17F antibodies, and heteroconjugate anti-IL-17F antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for IL-17F. The second binding target is any otherantigen, and advantageously is a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986). According toanother approach described in WO 96/27011, the interface between a pairof antibody molecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the CH3 region of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with IL-17Fsignaling. For example, cysteine residue(s) can be introduced into theFc region, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). (See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that hasdual Fc regions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines,” Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44; 201-208 (1984) describing useof MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S.Pat. No. 5,030,719, describing use of halogenated acetyl hydrazidederivative coupled to an antibody by way of an oligopeptide linker.Particularly preferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii)SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfa-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against IL-17F

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman WN “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, antibodies of the invention, which include amonoclonal antibody of the invention (e.g., a fully human monoclonalantibody), may be used as therapeutic agents. Such agents will generallybe employed to diagnose, prognose, monitor, treat, alleviate, and/orprevent a disease or pathology associated with IL-17F signaling in asubject. A therapeutic regimen is carried out by identifying a subject,e.g., a human patient suffering from (or at risk of developing) aninflammatory disease or disorder, using standard methods. An antibodypreparation, preferably one having high specificity and high affinityfor its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withthe signaling function of the target (e.g., IL-17F). Administration ofthe antibody may abrogate or inhibit or interfere with the binding ofthe target (e.g., IL-17F) with an endogenous ligand (e.g., IL-17R orIL-17RC) to which it naturally binds. For example, the antibody binds tothe target and modulates, blocks, inhibits, reduces, antagonizes,neutralizes, or otherwise interferes with IL-17F-induced proinflammatorycytokine production.

Diseases or disorders related to IL-17F signaling include autoimmunediseases or inflammatory diseases or disorders, including but notlimited to rheumatoid arthritis, Crohn's disease, psoriasis, multiplesclerosis chronic obstructive pulmonary disease, and asthma. IL-17F wasfound to be up-regulated in sputum of cystic fibrosis patients (seeMcAllister et al., J. Immunol. 175: 404-412 (2005)), and in the colon ofpatients suffering from inflammatory bowel disease (see Seiderer et al.,Inflamm. Bowel Dis. Dec. 18 2007, Epub. ahead of print). IL-17A/IL-17Fhas been shown to play a role in the recruitment of airway neutrophilia,suggesting a role in the pathogenesis of respiratory disease (see Lianget al., J. Immunol. 179(11): 7791-9 (2007)).

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barrè syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

IL-17F has also been shown to be upregulated by IL-21 signaling,suggesting that the pro-inflammatory effects associated with IL-21signaling are mediated by IL-17F, in addition to IL-17A and/or theIL-17F/IL17A complex (Wei et al., J Biol. Chem. 282(48):34605-10(2007)). As such, the antibodies of the invention are also useful fordiagnosing, prognosing, monitoring and/or treating disorders diseasesmediated by IL-21 signaling, including but not limited toinflammatory/autoimmune disorders such as inflammatory bowel disease,rheumatoid arthritis, transplant rejection, and psoriasis.

The huIL-17F antibodies of the invention are useful in treating,ameliorating, delaying the onset and/or progression or otherwisereducing the severity of an arthritic condition or one or more symptomsthereof. Prior to the instant invention, studies had determined thatIL-17F played little, if any role, in the development and/or progressionof autoimmune arthritic conditions. (See Ishigame et al., Immunity, vol.30: 108-119 (2009)). The experiments and data provided in the Examplesherein demonstrate the unexpected and surprising result that modulating,e.g., inhibiting, neutralizing or otherwise blocking, the expressionand/or activity of the IL-17F homodimer, but not the IL-17A/IL-17Fheterodimeric complex, significantly delayed the progression of thearthritic condition and reduced the level of inflammatory mediators suchas TNF-α, IL-6, IFN-γ, IL-1α, MCP-1 and IL-12/IL-23 (p40) incollagen-induced arthritis, an animal model for rheumatoid arthritis.

Accordingly, the invention includes methods of treating, ameliorating,delaying the onset and/or progression, and/or alleviating a symptom ofan arthritic condition by administering to a subject in need thereof ahuIL-17F antibody of the invention (e.g., 5E12, 41B10, 11C5, 21B10, 1F1,2E12, 5D3, 22F8, 28B11, 41A4 and 43G6) or an antibody that binds to thesame or similar epitope as a huIL-17F antibody of the invention. ThehuIL-17F antibody or antibody that binds to the same epitope as ahuIL-17F antibody of the invention is administered in an amountsufficient to treat, ameliorate, delay the onset, delay the progression,and/or alleviate one or more symptoms of the arthritic condition. Thesubject is, for example, human. In some embodiments, the arthriticcondition is an autoimmune arthritic condition. For example, in someembodiments, the arthritic condition is rheumatoid arthritis.

Symptoms associated with inflammatory-related disorders include, forexample, inflammation, fever, general malaise, fever, pain, oftenlocalized to the inflamed area, rapid pulse rate, joint pain or aches(arthralgia), rapid breathing or other abnormal breathing patterns,chills, confusion, disorientation, agitation, dizziness, cough, dyspnea,pulmonary infections, cardiac failure, respiratory failure, edema,weight gain, mucopurulent relapses, cachexia, wheezing, headache, andabdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation. Symptoms associated with immune-related disorders include,for example, inflammation, fever, loss of appetite, weight loss,abdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation, joint pain or aches (arthralgia), fatigue, rash, anemia,extreme sensitivity to cold (Raynaud's phenomenon), muscle weakness,muscle fatigue, changes in skin or tissue tone, shortness of breath orother abnormal breathing patterns, chest pain or constriction of thechest muscles, abnormal heart rate (e.g., elevated or lowered), lightsensitivity, blurry or otherwise abnormal vision, and reduced organfunction.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular inflammatory-relateddisorder. Alleviation of one or more symptoms of theinflammatory-related disorder indicates that the antibody confers aclinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

In another embodiment, antibodies directed against IL-17F and/or theIL-17F/IL17A complex may be used in methods known within the artrelating to the localization and/or quantitation of IL-17F or theIL-17A/IL-17F complex (e.g., for use in measuring levels of the IL-17Fand/or the IL-17A/IL-17F complex within appropriate physiologicalsamples, for use in diagnostic methods, for use in imaging the protein,and the like). In a given embodiment, antibodies specific to IL-17Fand/or the IL-17A/IL-17F complex, or derivative, fragment, analog orhomolog thereof, that contain the antibody derived antigen bindingdomain, are utilized as pharmacologically active compounds (referred tohereinafter as “Therapeutics”).

In another embodiment, an antibody specific for IL-17F and/or theheterodimeric IL-17A/IL-17F complex can be used to isolate an IL-17Fpolypeptide and/or a heterodimeric IL-17A/IL-17F complex polypeptide bystandard techniques, such as immunoaffinity, chromatography orimmunoprecipitation. Antibodies directed against the IL-17F proteinand/or the heterodimeric IL-17A/IL-17F complex (or a fragment thereof)can be used diagnostically to monitor protein levels in tissue as partof a clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

In yet another embodiment, an antibody according to the invention can beused as an agent for detecting the presence of IL-17F (or a proteinfragment thereof) in a sample. In some embodiments, the antibodycontains a detectable label. Antibodies are polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,F_(ab), scFv, or F_((ab)2)) is used. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Therapeutic Administration and Formulations of huIL-17F Antibodies

The antibodies of the invention (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, far example, in Remington's Pharmaceutical Sciences: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceuticallyacceptable carrier. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas autoimmune disorders and inflammatory diseases. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is preferably still detectableat effective concentrations at the site of treatment.

For example, the combination therapy can include one or more antibodiesof the invention coformulated with, and/or coadministered with, one ormore additional therapeutic agents, e.g., one or more cytokine andgrowth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostaticagents, as described in more detail below. Furthermore, one or moreantibodies described herein may be used in combination with two or moreof the therapeutic agents described herein. Such combination therapiesmay advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Preferred therapeutic agents used in combination with an antibody of theinvention are those agents that interfere at different stages in aninflammatory response. In one embodiment, one or more antibodiesdescribed herein may be coformulated with, and/or coadministered with,one or more additional agents such as other cytokine or growth factorantagonists (e.g., soluble receptors, peptide inhibitors, smallmolecules, ligand fusions); or antibodies or antigen binding fragmentsthereof that bind to other targets (e.g., antibodies that bind to othercytokines or growth factors, their receptors, or other cell surfacemolecules); and anti-inflammatory cytokines or agonists thereof.Nonlimiting examples of the agents that can be used in combination withthe antibodies described herein, include, but are not limited to,antagonists of one or more interleukins (ILs) or their receptors, e.g.,antagonists of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16,IL-18, IL-21 and IL-22; antagonists of cytokines or growth factors ortheir receptors, such as tumor necrosis factor (TNF), LT, EMAP-II,GM-CSF, FGF and PDGF. Antibodies of the invention can also be combinedwith inhibitors of, e.g., antibodies to, cell surface molecules such asCD2, CD3, CD4, CD8, CD20 (e.g., the CD20 inhibitor rituximab(RITUXAN®)), CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86(B7.2), CD90, or their ligands, including CD154 (gp39 or CD4OL), orLFA-1/ICAM-1 and VLA-4NCAM-1 (Yusuf-Makagiansar et al. (2002) Med. Res.Rev. 22:146-67). Preferred antagonists that can be used in combinationwith the antibodies described herein include antagonists of IL-1, IL-12,TNFα, IL-15, IL-18, and IL-22.

Examples of those agents include IL-12 antagonists, such as chimeric,humanized, human or in vitro-generated antibodies (or antigen bindingfragments thereof) that bind to IL-12 (preferably human IL-12), e.g.,the antibody disclosed in WO 00/56772; IL-12 receptor inhibitors, e.g.,antibodies to human IL-12 receptor; and soluble fragments of the IL-12receptor, e.g., human IL-12 receptor. Examples of IL-15 antagonistsinclude antibodies (or antigen binding fragments thereof) against IL-15or its receptor, e.g., chimeric, humanized, human or in vitro-generatedantibodies to human IL-15 or its receptor, soluble fragments of theIL-15 receptor, and IL-15-binding proteins. Examples of IL-18antagonists include antibodies, e.g., chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof), tohuman IL-18, soluble fragments of the IL-18 receptor, and IL-18 bindingproteins (IL-18BP). Examples of IL-1 antagonists includeInterleukin-1-converting enzyme (ICE) inhibitors, such as Vx740, IL-1antagonists, e.g., IL-1RA (anikinra, KINERET™, Amgen), sIL1RII(Immunex), and anti-IL-1 receptor antibodies (or antigen bindingfragments thereof).

Examples of TNF antagonists include chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof) to TNF(e.g., human TNFα), such as (HUMIRA™, D2E7, human TNFα antibody),CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Pharmacia), cA2 (chimeric anti-TNFα antibody; REMICADE®,Centocor); anti-TNF antibody fragments (e.g., CPD870); soluble fragmentsof the TNF receptors, e.g., p55 or p75 human TNF receptors orderivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusionprotein, ENBREL™; Immunex), p55 kdTNFR-IgG (55 kD TNF receptor-IgGfusion protein (LENERCEP®); enzyme antagonists, e.g., TNFα convertingenzyme (TACE) inhibitors (e.g., an alpha-sulfonyl hydroxamic acidderivative, and N-hydroxyformamide TACE inhibitor GW 3333, -005, or-022); and TNF-bp/s-TNFR (soluble TNF binding protein). Preferred TNFantagonists are soluble fragments of the TNF receptors, e.g., p55 or p75human TNF receptors or derivatives thereof, e.g., 75 kdTNFR-IgG, andTNFα converting enzyme (TACE) inhibitors.

In other embodiments, the antibodies described herein may beadministered in combination with one or more of the following: IL-13antagonists, e.g., soluble IL-13 receptors (sIL-13) and/or antibodiesagainst IL-13; IL-2 antagonists, e.g., DAB 486-IL-2 and/or DAB 389-IL-2(IL-2 fusion proteins, Seragen), and/or antibodies to IL-2R, e.g.,anti-Tac (humanized anti-IL-2R, Protein Design Labs). Yet anothercombination includes antibodies of the invention, antagonistic smallmolecules, and/or inhibitory antibodies in combination with nondepletinganti-CD4 inhibitors (DEC-CE9.1/SB 210396; nondepleting primatizedanti-CD4 antibody; IDEC/SmithKline). Yet other preferred combinationsinclude antagonists of the costimulatory pathway CD80 (B7.1) or CD86(B7.2), including antibodies, soluble receptors or antagonistic ligands;as well as p-selectin glycoprotein ligand (PSGL), anti-inflammatorycytokines, e.g., IL-4 (DNAX/Schering); IL-10 (SCH 52000; recombinantIL-10 DNAX/Schering); IL-13 and TGF-β, and agonists thereof (e.g.,agonist antibodies).

In other embodiments, one or more antibodies of the invention can becoformulated with, and/or coadministered with, one or moreanti-inflammatory drugs, immunosuppressants, or metabolic or enzymaticinhibitors. Nonlimiting examples of the drugs or inhibitors that can beused in combination with the antibodies described herein, include, butare not limited to, one or more of: nonsteroidal anti-inflammatorydrug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam,piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroidssuch. as prednisolone; cytokine suppressive anti-inflammatory drug(s)(CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors ofpurine biosynthesis, folate antagonists (e.g., methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotatedehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for usein combination with the antibodies of the invention include NSAIDs,CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists(e.g., methotrexate).

Examples of additional inhibitors include one or more of:corticosteroids (oral, inhaled and local injection); immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g.,sirolimus (rapamycin RAPAMUNE™ or rapamycin derivatives, e.g., solublerapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); agents which interfere with signaling by proinflammatorycytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinaseinhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variantsthereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesteraseType IV inhibitor); phospholipase inhibitors, e.g., inhibitors ofcytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketoneanalogs); inhibitors of vascular endothelial cell growth factor orgrowth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor;and inhibitors of angiogenesis. Preferred therapeutic agents for use incombination with the antibodies of the invention are immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus(rapamycin) or rapamycin derivatives, e.g., soluble rapamycinderivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2inhibitors, e.g., celecoxib and variants thereof; and phospholipaseinhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g.,trifluoromethyl ketone analogs.

Additional examples of therapeutic agents that can be combined with anantibody of the invention include one or more of: 6-mercaptopurines(6-MP); azathioprine sulphasalazine; mesalazine; olsalazine;chloroquine/hydroxychloroquine (PLAQUENIL®); pencillamine;aurothiornalate (intramuscular and oral); azathioprine; coichicine;beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral);xanthines (theophylline, aminophylline); cromoglycate; nedocromil;ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosineagonists; antithrombotic agents; complement inhibitors; and adrenergicagents.

Nonlimiting examples of agents for treating or preventing arthriticdisorders (e.g., rheumatoid arthritis, inflammatory arthritis,rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis andpsoriatic arthritis), with which an antibody of the invention may becombined include one or more of the following: IL-12 antagonists asdescribed herein; NSAIDs; CSAIDs; TNFs, e.g., TNFα, antagonists asdescribed herein; nondepleting anti-CD4 antibodies as described herein;IL-2 antagonists as described herein; anti-inflammatory cytokines, e.g.,IL-4, IL-10, IL-13 and TGFα, or agonists thereof; IL-1 or IL-1 receptorantagonists as described herein; phosphodiesterase inhibitors asdescribed herein; Cox-2 inhibitors as described herein; iloprost:methotrexate; thalidomide and thalidomide-related drugs (e.g., Celgen);leflunomide; inhibitor of plasminogen activation, e.g., tranexamic acid;cytokine inhibitor, e.g., T-614; prostaglandin E1; azathioprine; aninhibitor of interleukin-1 converting enzyme (ICE); zap-70 and/or lckinhibitor (inhibitor of the tyrosine kinase zap-70 or lck); an inhibitorof vascular endothelial cell growth factor or vascular endothelial cellgrowth factor receptor as described herein; an inhibitor of angiogenesisas described herein; corticosteroid anti-inflammatory drugs (e.g.,SB203580); TNF-convertase inhibitors; IL-11; IL-13; IL-17 inhibitors;gold; penicillamine; chloroquine; hydroxychloroquine; chlorambucil;cyclophosphamide; cyclosporine; total lymphoid irradiation;antithymocyte globulin; CD5-toxins; orally administered peptides andcollagen; lobenzarit disodium; cytokine regulating agents (CRAs) HP228and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);prednisone; orgotein; glycosaminoglycan polysulphate; minocycline(MINOCIN®); anti-IL2R antibodies; marine and botanical lipids (fish andplant seed fatty acids); auranofm; phenylbutazone; meclofenamic acid;flufenamic acid; intravenous immune globulin; zileuton; mycophenolicacid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.Preferred combinations include one or more antibodies of the inventionin combination with methotrexate or leflunomide, and in moderate orsevere rheumatoid arthritis cases, cyclosporine.

Preferred examples of inhibitors to use in combination with antibodiesof the invention to treat arthritic disorders include TNF antagonists(e.g., chimeric, humanized, human or in vitro-generated antibodies, orantigen binding fragments thereof, that bind to TNF; soluble fragmentsof a TNF receptor, e.g., p55 or p75 human TNF receptor or derivativesthereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein,ENBREL™), p55 kD TNF receptor-IgG fusion protein; TNF enzymeantagonists, e.g., TNFα converting enzyme (TACE) inhibitors);antagonists of IL-12, IL-15, IL-18, IL-22; T cell and B cell-depletingagents (e.g., anti-CD4 or anti-CD22 antibodies); small moleculeinhibitors, e.g., methotrexate and leflunomide; sirolimus (rapamycin)and analogs thereof, e.g., CCI-779; cox-2 and cPLA2 inhibitors; NSAIDs;p38 inhibitors, TPL-2, Mk-2 and NFkb inhibitors; RAGE or soluble RAGE;P-selectin or PSGL-1 inhibitors (e.g., small molecule inhibitors,antibodies thereto, e.g., antibodies to P-selectin); estrogen receptorbeta (ERB) agonists or ERB-NFkb antagonists. Most preferred additionaltherapeutic agents that can be coadministered and/or coformulated withone or more antibodies of the invention include one or more of a solublefragment of a TNF receptor, e.g., p55 or p75 human TNF receptor orderivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusionprotein, ENBREL™); methotrexate, leflunomide, or a sirolimus (rapamycin)or an analog thereof, e.g., CCI-779.

Nonlimiting examples of agents for treating or preventing multiplesclerosis with which antibodies of the invention can be combined includethe following: interferons, e.g., interferon-alpha 1a (e.g., AVONEX™;Biogen) and interferon-1b (BETASERON™ Chiron/Berlex); Copolymer 1(Cop-1; COPAXONE™ Teva Pharmaceutical Industries, Inc.); hyperbaricoxygen; intravenous immunoglobulin; cladribine; TNF antagonists asdescribed herein; corticosteroids; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; cyclosporine A,methotrexate; 4-aminopyridine; and tizanidine. Additional antagoniststhat can be used in combination with antibodies of the invention includeantibodies to or antagonists of other human cytokines or growth factors,for example, TNF, LT, IL-1, IL-2, IL-6, EL-7, IL-8, IL-12 IL-15, IL-16,IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Antibodies as described hereincan be combined with antibodies to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. The antibodies of the invention may also be combined withagents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines as described herein, IL-1b converting enzymeinhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cellsignaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof, as described herein, and anti-inflammatory cytokines (e.g.IL-4, IL-10, IL-13 and TGF).

Preferred examples of therapeutic agents for multiple sclerosis withwhich the antibodies of the invention can be combined includeinterferon-β, for example, IFNβ-1a and IFNβ-1b; copaxone,corticosteroids, IL-1 inhibitors, TNF inhibitors, antibodies to CD40ligand and CD80, IL-12 antagonists.

Nonlimiting examples of agents for treating or preventing inflammatorybowel disease (e.g., Crohn's disease, ulcerative colitis) with which anantibody of the invention can be combined include the following:budenoside; epidermal growth factor; corticosteroids; cyclosporine;sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptorantagonists; anti-IL-1 monoclonal antibodies; anti-IL-6 monoclonalantibodies; growth factors; elastase inhibitors; pyridinyl-imidazolecompounds; TNF antagonists as described herein; IL-4, IL-10, IL-13and/or TGFβ cytokines or agonists thereof (e.g., agonist antibodies);IL-11; glucuronide- or dextran-conjugated prodrugs of prednisolone,dexamethasone or budesonide; ICAM-1 antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP10; T Cell Sciences, Inc.); slow-releasemesalazine; methotrexate; antagonists of platelet activating factor(PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of agents for treating or preventing psoriasis withwhich an antibody of the invention can be combined include thefollowing: corticosteroids; vitamin D₃ and analogs thereof; retinoiods(e.g., soriatane); methotrexate; cyclosporine, 6-thioguanine; Accutane;hydrea; hydroxyurea; sulfasalazine; mycophenolate mofetil; azathioprine;tacrolimus; fumaric acid esters; biologics such as Amevive, Enbrel,Humira, Raptiva and Remicade, Ustekinmab, and XP-828L; phototherapy; andphotochemotherapy (e.g., psoralen and ultraviolet phototherapycombined).

Nonlimiting examples of agents for treating or preventing inflammatoryairway/respiratory disease (e.g., chronic obstructive pulmonarydisorder, asthma) with which an antibody of the invention can becombined include the following: beta2-adrenoceptor agonists (e.g.,salbutamol (albuterol USAN), levalbuterol, terbutaline, bitolterol);long-acting beta2-adrenoceptor agonists (e.g., salmeterol, formoterol,bambuterol); adrenergic agonists (e.g., inhaled epinephrine andephedrine tablets); anticholinergic medications (e.g., ipratropiumbromide); Combinations of inhaled steroids and long-actingbronchodilators (e.g., fluticasone/salmeterol (Advair in the UnitedStates, and Seretide in the United Kingdom)) or. budesonide/formoterol(Symbicort)); inhaled glucocorticoids (e.g., ciclesonide,beclomethasone, budesonide, flunisolide, fluticasone, mometasone,triamcinolone); leukotriene modifiers (e.g., montelukast, zafirlukast,pranlukast, and zileuton); mast cell stabilizers (e.g., cromoglicate(cromolyn), and nedocromil); antimuscarinics/anticholinergics (e.g.,ipratropium, oxitropium, tiotropium); methylxanthines (e.g.,theophylline, aminophylline); antihistamines; IgE blockers (e.g.,Omalizumab); M₃ muscarinic antagonists (anticholinergics) (e.g.,ipratropium, tiotropium); cromones (e.g., chromoglicate, nedocromil);xanthines (e.g., theophylline); and TNF antagonists (e.g., infliximab,adalimumab and etanercept).

In one embodiment, an antibody of the invention can be used incombination with one or more antibodies directed at other targetsinvolved in regulating immune responses, e.g., transplant rejection.

Nonlimiting examples of agents for treating or preventing immuneresponses with which an antibody of the invention can be combinedinclude the following: antibodies against other cell surface molecules,including but not limited to CD25 (interleukin-2 receptor-a), CD11a(LFA-1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4 (CD80 (137.1), e.g., CTLA4Ig—abatacept (ORENCIA®)), ICOSL, ICOS and/or CD86 (B7.2). In yet anotherembodiment, an antibody of the invention is used in combination with oneor more general immunosuppressive agents, such as cyclosporin A orFK506.

In other embodiments, antibodies are used as vaccine adjuvants againstautoimmune disorders, inflammatory diseases, etc. The combination ofadjuvants for treatment of these types of disorders are suitable for usein combination with a wide variety of antigens from targetedself-antigens, i.e., autoantigens, involved in autoimmunity, e.g.,myelin basic protein; inflammatory self-antigens, e.g., amyloid peptideprotein, or transplant antigens, e.g., alloantigens. The antigen maycomprise peptides or polypeptides derived from proteins, as well asfragments of any of the following: saccharides, proteins,polynucleotides or oligonucleotides, autoantigens, amyloid peptideprotein, transplant antigens, allergens, or other macromolecularcomponents. In some instances, more than one antigen is included in theantigenic composition.

For example, desirable vaccines for moderating responses to allergens ina vertebrate host, which contain the adjuvant combinations of thisinvention, include those containing an allergen or fragment thereof.Examples of such allergens are described in U.S. Pat. No. 5,830,877 andpublished International Patent Application No. WO 99/51259, which arehereby incorporated by reference in their entireties, and includepollen, insect venoms, animal dander, fungal spores and drugs (such aspenicillin). The vaccines interfere with the production of IgEantibodies, a known cause of allergic reactions. In another example,desirable vaccines for preventing or treating disease characterized byamyloid deposition in a vertebrate host, which contain the adjuvantcombinations of this invention, include those containing portions ofamyloid peptide protein (APP). This disease is referred to variously asAlzheimer's disease, amyloidosis or amyloidogenic disease. Thus, thevaccines of this invention include the adjuvant combinations of thisinvention plus Aβ peptide, as well as fragments of Aβ peptide andantibodies to Aβ peptide or fragments thereof.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto IL-17F and/or the heterodimeric IL-17A/IL-17F complex, the design ofother therapeutic modalities beyond antibody moieties is facilitated.Such modalities include, without limitation, advanced antibodytherapeutics, such as bispecific antibodies, immunotoxins, andradiolabeled therapeutics, generation of peptide therapeutics, genetherapies, particularly intrabodies, antisense therapeutics, and smallmolecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to IL-17F and/or the heterodimeric IL-17A/IL-17F complex,and another to a second molecule that are conjugated together, (ii) asingle antibody that has one chain specific to IL-17F and/or theheterodimeric IL-17A/IL-17F complex, and a second chain specific to asecond molecule, or (iii) a single chain antibody that has specificityto IL-17F and/or the heterodimeric IL-17A/IL-17F complex, and a secondmolecule. Such bispecific antibodies are generated using techniques thatare well known for example, in connection with (i) and (ii) See e.g.,Fanger et al. Immunol Methods 4:72-81 (1994) and Wright et al. Crit,Reviews in Immunol. 12125-168 (1992), and in connection with (iii) Seee.g., Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing IL-17F and/or the heterodimericIL-17A/IL-17F complex.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to IL-17F and/or theheterodimeric IL-17A/IL-17F complex and antibodies thereto, such as theantibodies of the invention or screening of peptide libraries,therapeutic peptides can be generated that are directed against IL-17Fand/or the heterodimeric IL-17A/IL-17F complex. Design and screening ofpeptide therapeutics is discussed in connection with Houghten et al.Biotechniques 13:412-421 (1992), Houghten PNAS USA 82:5131-5135 (1985),Pinalla et al. Biotechniques 13:901-905 (1992), Blake and Litzi-DavisBioConjugate Chem. 3:510-513 (1992). Immunotoxins and radiolabeledmolecules can also be prepared, and in a similar manner, in connectionwith peptidic moieties as discussed above in connection with antibodies.Assuming that the IL-17F and/or the heterodimeric IL-17A/IL-17F complexmolecule (or a form, such as a splice variant or alternate form) isfunctionally active in a disease process, it will also be possible todesign gene and antisense therapeutics thereto through conventionaltechniques. Such modalities can be utilized for modulating the functionof IL-17F and/or the heterodimeric IL-17A/IL-17F complex. In connectiontherewith the antibodies of the present invention facilitate design anduse of functional assays related thereto. A design and strategy forantisense therapeutics is discussed in detail in International PatentApplication No. WO 94/29444. Design and strategies for gene therapy arewell known. However, in particular, the use of gene therapeutictechniques involving intrabodies could prove to be particularlyadvantageous. See e.g., Chen et al. Human Gene Therapy 5:595-601 (1994)and Marasco Gene Therapy 4:11-15 (1997). General design of andconsiderations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the IL-17F and/or theheterodimeric IL-17A/IL-17F complex molecule and its interactions withother molecules in accordance with the present invention, such as theantibodies of the invention, and others can be utilized to rationallydesign additional therapeutic modalities. In this regard, rational drugdesign techniques such as X-ray crystallography, computer-aided (orassisted) molecular modeling (CAMM), quantitative or qualitativestructure-activity relationship (QSAR), and similar technologies can beutilized to focus drug discovery efforts. Rational design allowsprediction of protein or synthetic structures which can interact withthe molecule or specific forms thereof which can be used to modify ormodulate the activity of IL-17F, and/or the heterodimeric IL-17A/IL-17Fcomplex. Such structures can be synthesized chemically or expressed inbiological systems. This approach has been reviewed in Capsey et al.Genetically Engineered Human Therapeutic Drugs (Stockton Press, NY(1988)). Further, combinatorial libraries can be designed andsynthesized and used in screening programs, such as high throughputscreening efforts.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with binding of IL-17F and/or the heterodimericIL-17A/IL-17F complex to their innate receptor, or candidate or testcompounds or agents that modulate, block, inhibit, reduce, antagonize,neutralize or otherwise interfere with the signaling function of IL-17Fand/or the heterodimeric IL-17A/IL-17F complex. Also provided aremethods of identifying compounds useful to treat disorders associatedwith IL-17F and/or heterodimeric IL-17A/IL-17F complex signaling. Theinvention also includes compounds identified in the screening assaysdescribed herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which modulate the signaling function of IL-17F and/orthe heterodimeric IL-17A/IL-17F complex. The test compounds of theinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. (See, e.g., Lam, 1997. Anticancer Drug Design 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the signalingfunction of IL-17F and/or the heterodimeric IL-17A/IL-17F complex. Forexample, the antibody is monoclonal antibody 5E12 (“Mab05”) and theantigen is IL-17F and/or the heterodimeric IL-17A/IL-17F complex.

In another embodiment, the IL-17F homodimer is provided and exposed toat least one neutralizing monoclonal antibody. Formation of anantibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat disordersassociated with IL-17F signaling.

In another embodiment, a soluble protein of IL-17F is provided andexposed to at least one neutralizing monoclonal antibody. Formation ofan antibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat disordersassociated with IL-17F signaling.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a neutralizing antibody,such as for example monoclonal antibody 5E12, 41B10, 11C5, 21B10, 1F1,2E12, 5D3, 22F8, 28B11, 41A4, and 43G6, each of which modulates orotherwise interferes with proinflammatory cytokine production.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use soluble IL-17F, soluble IL-17A/IL-17F complex andfragments thereof.

In more than one embodiment; it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody (e.g. 5E12, 41B10, 11C5, 21B10, 1F1, 2E12, 5D3, 22F8, 28B11,41A4, and 43G6) or the antigen (e.g. the IL-17F protein or theIL-17A/IL-17F complex) can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated antibody or antigen molecules canbe prepared from biotin-NHS (N-hydroxy-succinimide) using techniqueswell-known within the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, other antibodies reactivewith the antibody or antigen of interest, but which do not interferewith the formation of the antibody-antigen complex of interest, can bederivatized to the wells of the plate, and unbound antibody or antigentrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immuno detection of complexes using such otherantibodies reactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic and Prophylactic Formulations

The huIL-17F MAbs of the invention are used in diagnostic andprophylactic formulations. In one embodiment, an IL-17F antagonist, suchas a huIL-17F MAb of the invention, is administered to patients that areat risk of developing one or more of the aforementioned autoimmune orinflammatory diseases, such as for example, without limitation,rheumatoid arthritis and other autoimmune arthritic conditions, Crohn'sdisease, psoriasis, multiple sclerosis chronic obstructive pulmonarydisease, asthma, osteoarthritis and cancer. A patient's or organ'spredisposition to one or more of the aforementioned autoimmune orinflammatory diseases can be determined using genotypic, serological orbiochemical markers.

In another embodiment of the invention, an IL-17F antagonist, such as ahulL-17F antibody is administered to human individuals diagnosed with aclinical indication associated with one or more of the aforementionedautoimmune or inflammatory diseases such as rheumatoid arthritis orother autoimmune arthritic conditions, Crohn's disease, psoriasis,multiple sclerosis chronic obstructive pulmonary disease, asthma,osteoarthritis, and cancer. Upon diagnosis, an IL-17F antagonist, suchas a huIL-17F antibody is administered to mitigate or reverse theeffects of the clinical indication associated with rheumatoid arthritisand other autoimmune arthritic conditions, Crohn's disease, psoriasis,multiple sclerosis chronic obstructive pulmonary disease, asthma,osteoarthritis and cancer.

Antibodies of the invention are also useful in the detection of IL-17Fand/or the heterodimeric IL-17A/IL-17F complex in patient samples andaccordingly are useful as diagnostics. For example, the huIL-17Fantibodies of the invention are used in in vitro assays, e.g., ELISA, todetect IL-17F and/or heterodimeric IL-17A/IL-17F complex levels in apatient sample.

In one embodiment, a huIL-17F antibody of the invention is immobilizedon a solid support (e.g., the well(s) of a microtiter plate). Theimmobilized antibody serves as a capture antibody for any IL-17F and/orany heterodimeric IL-17A/IL-17F complex that may be present in a testsample. Prior to contacting the immobilized antibody with a patientsample, the solid support is rinsed and treated with a blocking agentsuch as milk protein or albumin to prevent nonspecific adsorption of theanalyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of IL-17F and/or the heterodimeric IL-17A/IL-17F complexantigen in the test sample is determined by comparison with a standardcurve developed from the standard samples.

It will be appreciated that based on the results obtained using thehuIL-17F antibodies of the invention in an in vitro diagnostic assay, itis possible to stage a disease (e.g., a clinical indication associatedwith ischemia, an autoimmune or inflammatory disorder) in a subjectbased on expression levels of the IL-17F and/or the heterodimericIL-17A/IL-17F complex antigen. For a given disease, samples of blood aretaken from subjects diagnosed as being at various stages in theprogression of the disease, and/or at various points in the therapeutictreatment of the disease. Using a population of samples that providesstatistically significant results for each stage of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each stage is designated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Cloning, Expression and Purification of Human IL-17F, RatIL-17F, Cynomolgus IL-17F Cloning

The cDNAs encoding the mature human IL-17F (AF384857, aa 31-163) ratIL-17F (AAH91568, aa 21-153) and cynomolgus IL-17F (identical tosequence XP_(—)001106517 aa 31-163,) were amplified by PCR and cloned inPCR4TOPO vector (Invitrogen). Upon another PCR step, a His tag or a Histag followed by an AviTag (Avidity, Denver Colo.) were introduced at theN-terminus of the cytokine coding sequence. These constructs were thenfused to a leader sequence and sub-cloned in a corresponding expressionvectors.

Expression and Purification of Human IL-17F and Rat IL-17F fromBaculovirus-Infected Cells

His-tagged huIL-17F or rat IL-17F preceded by the GP67 leader sequence(MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) (SEQ ID NO: 100) weresub-cloned into a baculovirus bacmid vector pFASTBAC Dual (Invitrogen).After transfection into Sf9 cells, recombinant virus was isolated andamplified. For protein production, Hi5 cells or SF9 cells were infectedwith baculovirus and incubated at 27° C. for 3 days. Cell culture mediumwas cleared by centrifugation, filtered and concentrated about 10 timesin SartoFlow Slice 200 (Sartorius—Hydrosart cutoff 10 kD). Afteradjustment of pH to 7.0 and another centrifugation step, theconcentrated protein was purified using standard procedures on Ni-NTASuperflow columns (Qiagen) or HiTrap Chelating HP columns (GEHealthcare) charged with Ni²⁺ ions. IL-17F containing fractions werepooled and desalted on PD-10 columns (GE Healthcare).

Human IL-17F and rat IL-17F from baculovirus-infected cells wereessentially free of contaminants after one purification step, andappeared predominantly as disulfide-linked homodimers as demonstrated bynon-reducing SDS-PAGE. The biological activity of the His-tagged,baculovirus-expressed human IL-17F was comparable to the activity ofcommercial cytokines (E. Coli expressed huIL-17F, Peprotech EC or R&DSystems)

Expression and Purification of Human IL-17F and Rat IL-17F from CHOK1SVCells

huIL-17F or rat IL-17F coding sequences preceded by the CD33 leadersequence (MPLLLLLPLLWAGALAMD; SEQ ID NO:101), plus a His tag, and anAviTag (Avidity, Denver Colo.) were placed under the control of the hCMVpromoter in the expression vector pEE14.4. IL-17F was expressed from abicistronic mRNA containing a viral internal ribosome entry site (IRES)and the GFP coding sequence as the second cistron. The pEE14.4.vectorcontains the glutamine synthetase (GS) gene, essential for the survivalof transfected cells in selection medium containing methioninesulphoximine (MSX). Stable transfectants were generated in the CHOK1 SVcell line, property of Lonza Biologics. After four weeks of culture inthe presence of MSX high-expressing clones were identified, expanded andused for the production of human or rat IL-17F.

CHOK1SV—expressed human IL-17F and rat IL-17F were purified by Ni²⁺affinity chromatography. They were essentially free of contaminants andappeared as disulfide-linked homodimers on non-reducing SDS-PAGE gels.The biological activity of the His+Avi-tagged, CHO-expressed humanIL-17F was significantly decreased as compared to the activity of thecommercial huIL-17F, probably due to the presence of a bulky, double tagat the N-terminus.

Expression and Purification of Human IL-17F cnIL-17F from PEAK Cells

His-tagged huIL-17F or cnIL-17F coding sequences were fused to theGaussia princeps luciferase leader sequence (AF015993) and placed underthe control of the EF1 promoter in the episomal expression vector pEAK8.The cytokine-coding sequence was followed by a viral internal ribosomeentry site (IRES) and a second cistron (GFP). The pEAK8 vector containsthe puromycin resistance gene, the EBV nuclear antigen 1 (EBNA1) and theoriP origin of replication. EBNA1 and oriP are necessary for thepropagation of the pEAK8 vector as episomal DNA in human cells and thegeneration of stable transfectants. Stably transfected cells wereobtained after 7-10 days of culture in the presence of 2 ug/mL ofpuromycine. The populations of puromycine resistant cells were expandedand used for cytokine production.

PEAK—expressed purified by Ni²⁺ affinity chromatography were >95% pureand were found predominantly in the form of disulfide-linked homodimers,as demonstrated by non-reducing SDS-PAGE. The biological activity of theHis-tagged, PEAK-expressed human IL-17F was similar to the activity ofthe huIL-17F from commercial sources.

Example 2 Immunizations

Fully human monoclonal antibodies were generated using transgenicstrains of mice in which mouse antibody gene expression was suppressedand replaced with human antibody gene expression. Three strains oftransgenic mice were used:

-   -   1) HuMab® mouse (Medarex, Princeton N.J.)    -   2) KM™ mouse, a crossbred between HuMAb Mouse and Kirin's TC        Mouse (Kirin Pharma Company, Japan)    -   3) KM (FCγRIIb-KO) mouse, a strain derived from KM™ mouse, in        which the gene Fcgr2b coding for the inhibitory Fc gamma        Receptor IIB has been inactivated.

Mice were immunized either with human IL-17F or both human IL-17F andrat IL-17F. Two forms of antigen were used for immunizations:non-conjugated IL-17F or IL-17F conjugated to Keyhole Limpet Hemocyanin(KLH). Immunization strategies followed standard protocols from theliterature.

Sera of immunized animals were screened periodically by ELISA for thepresence of human IgG directed against huIL-17F and rat IL-17F. Most ofthe animals developed high-titer responses to human IL-17F. When bothrat IL-17F and huIL-17 were used for immunizations, most of the animalsdeveloped high-titer responses to both antigens. Antibodiescross-reactive to huIL-17A were sporadically generated in KM and KM(FCγRIIb-KO) mice immunized with huIL-17F as the only antigen (i.e.,without rat IL-17F). Contrary to the KM and KM (FCγRIIb-KO) mice, HuMAbmice did not develop cross-reactive titers to IL-17A, irrespective ofthe immunization protocol employed.

Example 3 Generation of Hybridomas

Fusion of Lymph Node Cells with SP2/0 Myeloma Cells

To obtain hybridomas, popliteal, inguinal, para-aortic, submandibular,cervical, axial, and brachial lymph nodes were removed from the mice anddigested with collagenase and DNAse. Single cells suspension of lymphnode cells was mixed at 1:1 ratio with SP2/0 myeloma cells and suspendedin Cytofusion Low Conductivity Medium (CPS-LCMC, CytoPulse Sciences,Inc.). Fusions were done with 30 to 60 million splenocytes in theCytoPulse CEEF50 Electrofusion apparatus as indicated by themanufacturer (Cyto Pulse Sciences, Inc). After electrofusion, cells wereincubated for approximately 1 hour at 37° C. to allow recovery beforedistributing into 96-well plates.

Culture of Hybridomas

Fused cells were resuspended in HAT selection medium and plated in 44 to52 96-well plates at a cell concentration of 0.1−0.2×10⁵ splenocytes perwell in 200 μl medium. Hybridoma selection proceeded for 14 days. Fusionof lymph nodes of immunized mice resulted in the generation ofhybridomas producing antibodies specific to huIL-17F or cross-reactiveantibodies specific to both huIL-17F and IL-17A.

Hybridoma Screening

Fourteen days after the fusion, hybridoma-containing plates werescreened for the presence of human IgG binding to human IL-17F and/orhuman IL-17A by FLISA (Fluorescence-Linked Immunosorbent Assay). Inbrief, 6 micron beads (Polybeads, cat. No 07312, Polysciences Inc.) werecoated with huIL-17F (both from Peprotech EC) or BSA (Sigma) and weredistributed into FMAT® 384-well optical plates (Applied Biosystems) at adensity of 5,000 beads per well. The beads were mixed with a smallvolume of hybridoma culture supernatants (30 μl per well) and incubatedovernight before addition of goat anti-human IgG Fc (JacksonImmunoresearch No 109-005-098) conjugated to FMAT Blue® dye (AppliedBiosystems). After an incubation period of 2 to 8 hours the fluorescenceof the beads was measured in an 8200 Cellular Detection System analyzer(Applied Biosystems). Hybridomas producing human IgGs that bound tohuIL-17F, but not to BSA, were expanded and subjected to furtheranalysis.

Example 4 Recombinant Antibody Generation

Antibody Sequence Cloning from Hybridoma

To isolate antibody variable heavy and light chain sequences, RNA wasfirst extracted from selected hybridoma and subjected to reversetranscription. Then, VH and VL sequences were amplified by PCR, clonedand further analyzed by DNA sequencing. In brief, template RNA wasextracted from hybridoma using RNeasy Plus kit (QIAGEN) and cDNA weregenerated using ready-to-go you-prime first-strand beads (GE Healthcare,No 27-9264-01) with oligo dT primers for the reverse transcription. VHand VL sequences were then amplified by PCR using primer setsrecognizing families of human variable heavy and light chains. Amplifiedsequences were cloned into pCR®4 vector using TOPO® TA Cloning Kit forSequencing (Invitrogen). Selected clones were then subject to DNAsequencing.

Antibody Reformatting, Germlining and Expression

Variable heavy and light chain sequences were reformatted into mammalianexpression vectors for antibody production and characterization. Inbrief, sequence analysis was performed with isolated VH and VL todetermine their germline. Due to primer mismatches, mutations wereintroduced in 5′ of 1F1 and 11C5 VLs during the PCR amplification step.Therefore, site directed mutagenesis, conducted with the Quikchange kit(Strategene), was used to convert these mutations back to the Humangermline sequence. Afterward, VH and VL sequences were sub-cloned intomammalian expression system in frame of Human IgG1 and IgKappabackbones. Antibody heavy and light chains corresponding vectors werethen transfected in PEAK cells using TransIT®-LT1 transfection reagent(Mirus Bio) and cultured in DMEM+IgG-depleted serum+Glutamine.PEAK-expressed antibodies were then purified from the supernatant usingMabSelectSure slurry (GE Healthcare).

Example 5 Cross-Reactivity of huIL-17F Antibodies

Binding assay: huIL-17F antibodies were tested for their ability to bindto the other members of the IL-17 family of cytokines, as well as toIL-17A and IL-17F from other species. The assay was performed in theFLISA format, as described above. The following recombinant cytokineswere bound to polystyrene beads and tested for their ability to bindhuIL-17F antibodies: huIL17B (PeprotechEC, cat No 200-28), huIL-17C (R&DSystems, cat No), huIL-17D (PeprotechEC, cat No 200-27), huIL-17E(huIL-25, PeprotechEC, cat No 200-24), muIL-17A (PeprotechEC, cat No210-17), muIL-17F (PeprotechEC, cat No 200-17F), rat IL-17F (His-tagged,produced in house in insect cells), rat IL-17A (His-tagged, produced inhouse in PEAK cells), cyIL-17F (His-tagged, produced in house in PEAKcells), and cyIL-17A (His-tagged, produced in house in PEAK cells). Theability of the individual the huIL-17F antibodies to bind thesedifferent cytokines is summarized in Table 3 below:

TABLE 3 Cross-reactivity of huIL-17F antibodies as determined by FLISA(n.t. = not tested) species human cynomolgus mouse rat dimer: clone IL-IL- IL- IL- name IL-17F IL-17A 17A/F IL-17B IL-17C IL-17D IL-17E IL-17FIL-17A 17A/F IL-17F IL-17A 17A/F IL-17F 17A 5E12 + − − − − − − + − − − −− − − 41B10 + − + − − − − + − + − − − − − 11C5 + − + − − − − + − + − − −− − 21B10 + − + − − − − + − + − − − − − 1F1 + − + − − − − + − + − − − −− 2E12 + − + − − − − + − + − − − − − 5D3 + − + − − − − + − + − − − − −22F8 + − + − − − − + − + − − − − − 28B11 + − + − − − − + − + − − − − −41A4 + − + − − − − + − +/− − − − − − 43G6 + − + − − − − + − + − − − − −

Example 6 Neutralization Potency of huIL-17F Antibodies IL-6 Secretionby IL-17-Stimulated Mouse Embryonic Fibroblasts

Human and cynomolgus IL-17A and IL-17F bind the corresponding mouseIL-17 receptor complex. As a consequence, mouse fibroblasts respond tohuman or cynomolgus IL-17A and IL-17F by secreting IL-6. Co-stimulationwith mouse TNF was shown to synergize with IL-17 signaling (Ruddy et al.2004, J. Biol. Chem. 279:2559) significantly increasing the sensitivityof the mouse fibroblasts to IL-17 cytokines. Mouse C57BL/6 embryonicfibroblasts (MEF, ATCC No SCRC-1008) were therefore used to assay forthe neutralization capacity of hull-17F antibodies to neutralizehulL-17F, cyIL-17F, huIL-17A/F heterodimer and cyIL-17A/F heterodimerbiological activity.

Briefly, MEF cells seeded in 96-well plates in DMEM+Glutamine+10% FetalBovine Serum (PBS) were cultured for 48 h before the addition of IL-17cytokines and mouse TNFα at 10 ng/ml (Peprotech EC, cat No 315-01A). Inassays for MAb neutralizing activity, the IL-17 cytokines werepre-incubated with the antibody for 1 hour before adding to the cells.After 24 hours of stimulation in the presence of human or cynomolgusIL-17A/F heterodimers (50 ng/ml) or after 40 hours of stimulation in thepresence of human or cynomolgus IL-17F (5 ng/ml), supernatants werecollected and the concentration of mouse IL-6 was measured by sandwichELISA using rat anti mouse IL6 antibody (BD cat No 554400) for captureand a second, biotinylated, rat anti mouse IL6 antibody (BD 554402) plusstreptavidin HRP (Jackson Immunoresearch 016-030-084) for detection. Noinhibition of huIL-17A/F heterodimer or cyIL-17A/F heterodimer wasobserved with any of the anti IL-17F antibodies tested. The values ofIC₅₀ obtained with human and cynomolgus IL-17F homodimers are summarizedin Table 4 below and were obtained from IL-6 calibration curves usingstandard statistical techniques.

TABLE 4 Neutralization potency (IC₅₀ values) of huIL- 17F antibodies inMEF cells stimulated with huIL-17F or cy IL-17F homodimers and mTNF-αspecies human cynomolgus IL-17 dimer: IL-17F IL-17F IL-17 conc. 5 ng/ml5 ng/ml clone name IC50 (nM) 5E12 1.8 22 41B10 3.9 18 11C5 0.4 2.9 21B102.0 8.7 1F1 1.1 11 2E12 5.9 26 5D3 0.5 12 22F8 2.0 10 28B11 1.2 6.3 41A40.6 7.2 43G6 1.1 8.4

Example 7 Experimental Model of Disease: Collagen-Induced Arthritis(CIA)

IL-17A plays an important role in the pathogenesis of arthritis,promoting the release of mediators of inflammation and cartilagedestruction. The neutralization of IL-17A has been demonstrated toattenuate arthritis in various experimental models, including CIA(Collagen-Induced Arthritis). Given that IL-17F is closely related toIL-17A, and the fact that it is overexpressed in the synovia ofrheumatoid arthritis (RA) patients, the effect of neutralizing thiscytokine was explored in a model of RA. To this goal, an anti mIL-17F(mouse IL-17F) antibody that potently neutralized mouse IL-17Fhomodimers—but not IL-17A/F heterodimers—was generated, and the effectsof this anti-mIL-17F antibody in the CIA animal model for RA weretested.

In brief, 8-10 week-old male DBA-1J mice were immunized with 100micrograms bovine collagen type II in Complete Freund Adjuvant (CFA).Collagen type II in CFA was injected intradermally at the base of thetail. Three weeks later, 100 micrograms of collagen type II inIncomplete Freund Adjuvant (IFA) was injected intradermally to inducedisease. The first signs of disease usually appeared 4 to 10 days afterthe collagen-IFA boost. Animals that started to develop arthritis wererecruited to the study and distributed into the following treatmentgroups:

-   -   1) Isotype control (mouse IgG1 k), two times per week for three        weeks at 300 micrograms per injection (n=10)    -   2) Hamster murine chimeric anti TNF-alpha, once a week for three        weeks at 300 micrograms per injection (n=10)    -   3) Anti mouse IL-17F antibody (mouse IgG1k) two times per week        for three weeks at 300 micrograms per injection (n=11)

The three treatment groups were balanced to contain equivalent number ofanimals recruited at different clinical severity scores (1 to 3).Clinical scoring of the disease was performed three times per week usingstandard arthritis scoring methods. Scoring was 0-4 per paw (where 0means no disease and 4 represents edema that involves the entire paw)with a theoretical maximum cumulative score of 16 scores per animal. Inaddition to clinical severity scoring, serum levels of keyproinflammatory cytokines were determined by Luminex at termination (day22 after recruitment).

The progression of clinical scores and the cytokine serum levels areshown in FIGS. 1 and 2, respectively. The neutralization of IL-17Fhomodimers was sufficient to significantly delay the progression of thedisease and reduced the levels of inflammatory mediators. These findingssuggest that IL-17F is a candidate target for the therapy of autoimmunediseases such as rheumatoid arthritis.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the descriptionand examples above are for purposes of illustration and not limitationof the following claims.

1. An isolated fully human monoclonal anti-IL-17F antibody, or fragmentthereof, wherein said antibody comprises: (a) a V_(H) CDR1 regioncomprising the amino acid sequence of SEQ ID NO: 45, 48, 51, 64, 67 or70; (b) a V_(H) CDR2 region comprising the amino acid sequence of SEQ IDNO: 46, 49, 52, 54, 62, 65, 68, 71 or 73; (c) a V_(H) CDR3 regioncomprising the amino acid sequence of SEQ ID NO: 47, 50, 53, 55, 63, 66,69 or 72; (d) a V_(L) CDR1 region comprising the amino acid sequence ofSEQ ID NO: 74, 77, 80, 85, 91 or 94; (e) a V_(L) CDR2 region comprisingthe amino acid sequence of SEQ ID NO: 75, 78, 81, 83, 86, 92 or 96; and(f) a V_(L) CDR3 region comprising the amino acid sequence of SEQ ID NO:76, 79, 82, 84, 93, 95, 97, 98 and 99, wherein said antibody bindsIL-17F.
 2. The antibody of claim 1, wherein said antibody does not bindthe IL-17A homodimer.
 3. The antibody of claim 1, wherein said antibodyis an IgG isotype.
 4. The antibody of claim 1, wherein said antibody isan IgG1 isotype.
 5. The antibody of claim 1, wherein said antibodycomprises a heavy chain variable sequence comprising an amino acidsequence selected from SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38 and 42and a light chain variable sequence comprising the amino acid sequenceselected from SEQ ID NO: 12, 16, 20, 24, 28, 32, 36, 40 and
 44. 6. Anisolated fully human monoclonal antibody comprising a heavy chainvariable sequence comprising the amino acid sequence of SEQ ID NO: 10,14, 18, 22, 26, 30, 34, 38 and 42 and a light chain variable sequencecomprising the amino acid sequence of SEQ ID NO: 12, 16, 20, 24, 28, 32,36, 40 and 44, wherein said antibody binds IL-17F.
 7. The antibody ofclaim 6, wherein said antibody does not bind the IL-17A homodimer. 8.The antibody of claim 7, wherein said antibody is an IgG isotype.
 9. Apharmaceutical composition comprising the antibody of claim 1 and acarrier.
 10. A pharmaceutical composition comprising the antibody ofclaim 6 and a carrier.
 11. A method of alleviating a symptom of aclinical indication associated rheumatoid arthritis, Crohn's disease,psoriasis, multiple sclerosis chronic obstructive pulmonary disease, orasthma in a subject, the method comprising administering an antagonistof IL-17F to a subject in need thereof in an amount sufficient toalleviate the symptom of the clinical indication associated withrheumatoid arthritis, Crohn's disease, psoriasis, multiple sclerosischronic obstructive pulmonary disease, or asthma.
 12. The method ofclaim 11, wherein said subject is a human.
 13. The method of claim 11,wherein said antagonist is a monoclonal antibody or fragment thereof.14. The method of claim 13, wherein said monoclonal antibody is anantibody according claim 1 or fragment thereof.
 15. A method ofalleviating a symptom of an autoimmune disease or inflammatory disorder,the method comprising administering an antibody according to claim 1 toa subject in need thereof in an amount sufficient to alleviate thesymptom of the autoimmune disease or inflammatory disorder in thesubject.
 16. The method of claim 15, wherein said subject is a human.17. A method of alleviating a symptom of an arthritic condition, themethod comprising administering an antibody according to claim 1 to asubject in need thereof in an amount sufficient to alleviate the symptomof the arthritic condition in the subject.
 18. The method of claim 17,wherein said subject is a human.
 19. The method of claim 17, wherein thearthritic condition is an autoimmune arthritic condition.
 20. The methodof claim 17, wherein the arthritic condition is rheumatoid arthritis.